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Metabolic Glycan Imaging by Isonitriletetrazine Click Chemistry

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Metabolic Glycan Imaging by Isonitriletetrazine Click Chemistry CHEMBIOCHEM COMMUNICATIONS DOI: 10.1002/cbic.201300130 Metabolic Glycan Imaging by Isonitrile–Tetrazine Click Chemistry Shaun Stairs,[a] Andr A. Neves,[b] Henning Stçckmann,[a] Yelena A. Wainman,[a] Heather Ireland-Zecchini,[b] Kevin M. Brindle,[b] and Finian J. Leeper*[a] Bioorthogonal chemistry is the specific and exclusive reaction electrophiles such as maleimides, but the thiol group is also between a probe and a reporter group in the presence of the not truly bioorthogonal and is present on many biomolecules. varied functionality present in biological systems.[1,2] In the An unactivated alkene reacts very slowly with tetrazines,[18] but chemical-reporter strategy, a biologically inert functional group a cyclopropene group reacts considerably faster and has re- is incorporated into the desired biomolecule, and, at the ap- cently been successfully incorporated into cell-surface glycans, propriate time, the reaction with a complementary abiotic initially by using a cyclopropene-conjugated sialic acid[19] but functionality linked to various probes allows specific labelling very recently also with an N-acyl-mannosamine.[20] The azide of the biomolecule.[3] This strategy has been used to label vari- functional group has been most useful for these types of stud- ous biomolecules including proteins, lipids and glycans.[4–7] ies as it is not found in mammals and it undergoes a specific Glycans are complex biological macromolecules consisting 1,3-dipolar cycloaddition with alkynes, either catalysed by of covalently linked scaffolds of monosaccharide units. Most copper or promoted by ring strain. The azide group has also glycans exist as membrane-bound glycoconjugates, but many been used for metabolic labelling of bacterial lipopoly- are secreted and become part of the extracellular matrix. They saccharides.[23] New bioorthogonal chemistries that are com- have important roles in intercellular signalling, cell adhesion patible with the metabolic glycan labelling strategy and are and motility, and have also been shown to play a pivotal role additionally orthogonal to the azide reporter group, are ur- in cancer onset and progression.[8] In many cancer phenotypes, gently required to further facilitate the study of cell-surface certain sugars, particularly sialic acid, are overexpressed, thus glycosylation. glycan imaging has become an attractive target for clinical de- The isonitrile (or isocyano) group is neutral overall and con- velopment.[9,10] We have recently demonstrated that glycan sists of just two atoms. Isonitriles are stable at biologically rele- imaging can be used to detect tumours in vivo.[11] vant pH values and display no appreciable toxicity in mam- Labelling of glycoproteins in living systems presents a partic- mals.[24–26] Like the widely utilised azide group, isonitriles are ular challenge as the chemical reporter must be introduced not found in humans, though they are found in some natural metabolically. This is achieved by feeding to the cells a sugar products from various marine and terrestrial sources.[27,28] Isoni- which is a metabolic precursor with an additional chemical re- triles undergo a [4+1] cycloaddition with tetrazines, which porter group attached. This sugar is then incorporated by the have already been used extensively as bioorthogonal reaction intracellular machinery into the growing glycan. The size and partners for trans-cyclooctenes.[29,30] With many primary and charge of the chemical reporter are of key importance if the secondary isonitriles, a tautomerisation to an imine occurs, and precursor is to be accepted by the host cell’s enzymatic path- in aqueous solution this imine hydrolyses rapidly. However, we ways. Metabolic glycan labelling has currently only been ach- have shown that adducts with tertiary isonitriles, which cannot ieved by using azide,[9,12–14] alkyne,[15] ketone,[16] thiol,[17] tautomerise, are remarkably stable and that, with a primary 3- alkene[18] and cyclopropene[19–21] groups as the chemical report- isocyanopropionyl ester, the imine tautomerises further to er group. The alkyne chemical reporter requires a copper cata- a conjugated enamine, which is slow to hydrolyse.[31] The rates lyst for ligation with an azide, thus its utility for live-cell experi- of reaction of a dipyridyltetrazine probe with primary (3-isocya- ments is limited, although biocompatible copper catalysts are nopropionate) and tertiary isonitriles were 0.12 and being developed.[22] Ketone group labelling is achieved by re- 0.57mÀ1 sÀ1, and the adducts have half-lives of 16 and action with a hydrazide probe; this presents two major prob- 63 hours, respectively, in aqueous solution.[31] Thus, in terms of lems: the reaction is not truly bioorthogonal, as ketones are reactivity and stability of the conjugate, the tertiary isonitrile found in many biomolecules, and reduced pH values are re- would be a more attractive choice for a bioorthogonal ligation, quired for efficient ligation. Thiosugars can be labelled with but its greater size might prevent its metabolic incorporation into glycans by the host cell’s enzymes. [a] S. Stairs, Dr. H. Stçckmann, Y. A. Wainman, Dr. F. J. Leeper We hypothesised that, owing to its small size, stability and Department of Chemistry, University of Cambridge reactivity with tetrazine, the isonitrile group would be an ideal Lensfield Road, Cambridge, CB2 1EW (UK) candidate for a chemical reporter suitable for glycan imaging. E-mail: [email protected] In this paper, we present a proof-of-principle study in which [b] Dr. A. A. Neves, Dr. H. Ireland-Zecchini, Prof. K. M. Brindle isonitrile-bearing sugars are incorporated into cell surfaces and Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE (UK) labelled by a bioorthogonal isonitrile–tetrazine click reaction. Supporting information for this article is available on the WWW under First we checked that both primary and tertiary isonitriles http://dx.doi.org/10.1002/cbic.201300130. are bioorthogonal by incubating them with 10 mm glutathione 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 0000, 00,1–5 &1& These are not the final page numbers! ÞÞ CHEMBIOCHEM COMMUNICATIONS www.chembiochem.org for 24 h at pH 7.4 and 378C. No reaction was detected by 1H NMR spectroscopy (see the Supporting Information). To begin the study itself, primary and tertiary isonitrile deriv- atives of glucosamine, galactosamine and mannosamine were synthesised (Scheme 1). Ac4GlcN-n-Iso and Ac4GalN-n-Iso were Scheme 2. A) Structure of the tetrazine-biotin conjugate. B) Comparison of the shapes of the -NAz and N-n-Iso groups Lewis lung carcinoma (LL2) cells were seeded at a density of 2104 cells cmÀ2 and incubated for 24 h. The cells were then exposed to either the vehicle (DMSO, <0.25% v/v) or isonitrile sugar at 200 mm and incubated for a further 24 h. The cells were harvested and treated first with 100 mm Tz–biotin for 30 min then, after a series of washes with ice-cold buffer, with neutravidin-DyLight680 for 15 min. After a final set of washes, Scheme 1. Structures of the six isonitrile sugars used in this study. the cells were analysed by flow cytometry (Figure 1). There was no significant difference in the mean fluorescence intensity (MFI) of the tertiary isonitrile sugar pulsed cells and synthesised by 1-ethyl-3-(3-(dimethylamino)propyl)carbodii- the control cells. As it has previously been shown that the click mide hydrochloride (EDC) coupling of the corresponding O- reaction between a similar tetrazine probe and a tertiary isoni- peracetylated, free-amine sugars with 3-formamido- propanoic acid followed by dehydration with phos- phorus oxychloride. Ac4ManN-n-Iso was produced by a similar coupling reaction between mannosamine and 3-(formamido)propanoic acid, followed by pera- cetylation with acetic anhydride and pyridine, and then dehydration of the formamide. The tertiary iso- nitrile sugars Ac4GlcN-t-Iso, Ac4GalN-t-Iso and Ac4ManN-t-Iso were formed in an analogous fashion by coupling 2-isocyano-2-methylpropanoic acid with the same amino sugar derivatives (see the Support- ing Information for details). A two-step labelling procedure is normally used to detect incorporation of chemical reporter groups into cell-surface glycans; the cell surface is initially biotinylated by treatment with the chemical reporter. The biotinylated surface can then be visualised by using fluorescently labelled neutravidin followed by confocal microscopy or flow cytometry. This ap- proach improves the signal-to-background ratio (SBR), as it allows one to compensate for the slow rate of the first bioorthogonal reaction by delivering a high concentration of non-fluorogenic probe. The fluorescent component can then be delivered at a much lower concentration, due to the rapid kinet- Figure 1. Flow cytometry results from the two-step labelling procedure. LL2 cells were ics of biotin–neutravidin binding, thus minimising exposed to either vehicle (DMSO, <0.25% v/v, control) or one of the isonitrile sugars. the background signal that arises from nonspecific The mean fluorescence intensity (MFI) of the cells after the labelling procedure is shown. binding. To apply this approach, a tetrazine-biotin Error bars represent standard deviation of three repeats, 10000 events per repeat (see Figures S1 and S2 for details). Numbers above bars are the SBRs (sugar MFI/control MFI) conjugate (Tz–biotin, Scheme 2A) was synthesised and the associated error. *P<0.05, **P<0.005, differences between control and sugar (see the Supporting Information). MFI were significant, two-tailed t-test. 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 0000, 00,1–5 &2& These are not the final page numbers! ÞÞ CHEMBIOCHEM COMMUNICATIONS www.chembiochem.org trile is bioorthogonal, the sugars had either not been delivered To confirm our findings, quantitative epifluorescence micros- to the cell surface as glycans or the isonitrile group had been copy images of labelled cells were obtained. As little labelling degraded in the process.
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